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Nonthyrotoxic prevention of diet-induced insulin resistance by 3,5-diiodo-L-thyronine in rats.

de Lange P, Cioffi F, Senese R, Moreno M, Lombardi A, Silvestri E, De Matteis R, Lionetti L, Mollica MP, Goglia F, Lanni A - Diabetes (2011)

Bottom Line: T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation.At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated.Proteomic analysis of the hepatic protein profile supported these changes.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy.

ABSTRACT

Objective: High-fat diets (HFDs) are known to induce insulin resistance. Previously, we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented gain in body weight and adipose mass. Here we investigated whether and how T2 prevented HFD-induced insulin resistance.

Research design and methods: We investigated the biochemical targets of T2 related to lipid and glucose homeostasis over time using various techniques, including genomic and proteomic profiling, immunoblotting, transient transfection, and enzyme activity analysis.

Results: Here we show that, in rats, HFD feeding induced insulin resistance (as expected), whereas T2 administration prevented its onset. T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation. At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated. Proteomic analysis of the hepatic protein profile supported these changes.

Conclusions: T2, by activating SIRT1, triggers a cascade of events resulting in improvement of the serum lipid profile, prevention of fat accumulation, and, finally, prevention of diet-induced insulin resistance.

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Related in: MedlinePlus

T2 shifts hepatic gene and protein expression profiles toward increased lipid handling and decreased lipogenesis and gluconeogenesis. A–H: Quantitative RT-PCR analysis showing that T2 modulates gene expression in favor of lipid reduction (A), increases expression of genes involved in mitochondrial biogenesis (B), increases PPARα/δ expression (C and F), and genes involved in hepatic fatty acid oxidation (D and G), and normalizes the expression of genes involved in glucose homeostasis (E and H), after 2 weeks (A–E) or 4 weeks (F–H) of HFD-T2 treatment. Expression was normalized to that of Cyclophilin F. I and J: Proteomic analysis revealed protein profile changes toward adjustment of hepatic glucose metabolism (I) and lipid metabolism (J) to the HFD challenge. Left: Quantification of the data (for each treatment, expressed relative to the value obtained for control [N] rats, which was set as 1.0). Right: Representative 2D-E subsections obtained from livers of N, HFD, and HFD-T2 (4 weeks) rats. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups; ***P < 0.05 vs. HFD-fed group. □, N; ■, HFD; ▨, HFD-T2.
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Figure 4: T2 shifts hepatic gene and protein expression profiles toward increased lipid handling and decreased lipogenesis and gluconeogenesis. A–H: Quantitative RT-PCR analysis showing that T2 modulates gene expression in favor of lipid reduction (A), increases expression of genes involved in mitochondrial biogenesis (B), increases PPARα/δ expression (C and F), and genes involved in hepatic fatty acid oxidation (D and G), and normalizes the expression of genes involved in glucose homeostasis (E and H), after 2 weeks (A–E) or 4 weeks (F–H) of HFD-T2 treatment. Expression was normalized to that of Cyclophilin F. I and J: Proteomic analysis revealed protein profile changes toward adjustment of hepatic glucose metabolism (I) and lipid metabolism (J) to the HFD challenge. Left: Quantification of the data (for each treatment, expressed relative to the value obtained for control [N] rats, which was set as 1.0). Right: Representative 2D-E subsections obtained from livers of N, HFD, and HFD-T2 (4 weeks) rats. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups; ***P < 0.05 vs. HFD-fed group. □, N; ■, HFD; ▨, HFD-T2.

Mentions: The effects of T2 through SIRT1 clearly involve inhibition of lipogenesis and increased mitochondrial activity, through deacetylation of SREBP-1c and PGC-1α. Acetylated SREBP-1c induces transcription of critical lipogenic genes, and we found that after 2 weeks in the HFD-T2 animals, the SREBP-1c target genes ACC and fatty acid synthase (FAS) were downregulated (Fig. 4A). A third gene involved in lipogenesis, Spot 14 (S14), was also downregulated by T2, and a lipolytic gene, hepatic lipase (HL), involved in downregulation of triglycerides, was upregulated (Fig. 4A). Regarding mitochondrial biogenesis, T2 deacetylated PGC-1α but did not increase PGC-1α gene expression, and neither that of nuclear respiratory factors 1 and 2 (NRF1 and NRF2). However, those of mitochondrial transcription factor A and cytochrome oxidase subunit IV were increased in the HFD-T2 animals (Fig. 4B). Because fatty acid oxidation is known to be governed by PPARα and PPARδ in the liver, we measured the expression of PPARα and PPARδ, as well as the expression of a number of known PPARα/δ target genes. PPARs were targets of both AMPK and SIRT1, and gene expression was measured at both the 2-week time point (when only SIRT1 activity was increased) and the 4-week time point (when both SIRT1 and AMPK activities were increased). The PPARα/δ target genes were as follows: CPT1a and CPT2 (each involved in mitochondrial fatty acid uptake), acyl-CoA oxidase (AOX; a key enzyme in peroxisomal fatty acid oxidation), uncoupling protein 2 (UCP2; a PPAR target gene that is not translated into protein in the liver), and mitochondrial thioesterase I (MTE I; involved in mitochondrial lipid handling). In addition, because glucose tolerance was significantly ameliorated in HFD-T2 rats, we measured the expression of key genes in glucose homeostasis, which have been shown to be targets of AMPK and SIRT1. These key genes were as follows: phosphoenolpyruvate carboxykinase (PEPCK; which converts oxaloacetate to phosphoenolpyruvate and carbon dioxide), liver pyruvate kinase (LPK; a glycolytic enzyme that converts phosphoenolpyruvate to pyruvate), and glucose-6-phosphatase (G6Pase; which converts glucose-6-phosphate to glucose, which is then released from the hepatocyte). At 2 weeks, HFD-T2 rats displayed (vs. HFD rats) the following: 1) increased expression of PPARα, but not of PPARδ (Fig. 4C); 2) significant upregulation of the expression of CPT1a and CPT2 (Fig. 4D); and 3) significant reductions in the expression of LPK and G6Pase (Fig. 4E). At 4 weeks (again vs. HFD animals), HFD-T2 animals displayed significantly increased expression of both PPARα and PPARδ (Fig. 4F) and of all the above-mentioned PPARα/δ target genes (Fig. 4G). The expression of LPK and G6Pase were downregulated (as at 2 weeks), and that of PEPCK was still unaltered by T2 (Fig. 4H). Subsequently, we performed a high-resolution differential proteomic analysis on livers from N, HFD, and HFD-T2 rats. From the spots showing differential expression among the three analyzed groups, nine protein spots were identified and selected as proteins involved in glucose or lipid metabolism (Fig. 4I and J and Table 1). T2 treatment 1) prevented the induced elevations in glycolytic (LPK) and gluconeogenic (PEPCK, fructose-1,6-bisphosphatase 1 [FBPase1], and isocitrate dehydrogenase [IDH]) enzymes (Fig. 4I and Table 1), 2) had a strong positive impact on the expression of enzymes involved in fatty acid oxidation (enoyl CoA hydratase [ECH]), and 3) had normalizing effects on lipogenic proteins (carbonic anhydrase 3 [CA3] and glycerol-3-phosphate dehydrogenase [GPDH]) (Fig. 4J and Table 1).


Nonthyrotoxic prevention of diet-induced insulin resistance by 3,5-diiodo-L-thyronine in rats.

de Lange P, Cioffi F, Senese R, Moreno M, Lombardi A, Silvestri E, De Matteis R, Lionetti L, Mollica MP, Goglia F, Lanni A - Diabetes (2011)

T2 shifts hepatic gene and protein expression profiles toward increased lipid handling and decreased lipogenesis and gluconeogenesis. A–H: Quantitative RT-PCR analysis showing that T2 modulates gene expression in favor of lipid reduction (A), increases expression of genes involved in mitochondrial biogenesis (B), increases PPARα/δ expression (C and F), and genes involved in hepatic fatty acid oxidation (D and G), and normalizes the expression of genes involved in glucose homeostasis (E and H), after 2 weeks (A–E) or 4 weeks (F–H) of HFD-T2 treatment. Expression was normalized to that of Cyclophilin F. I and J: Proteomic analysis revealed protein profile changes toward adjustment of hepatic glucose metabolism (I) and lipid metabolism (J) to the HFD challenge. Left: Quantification of the data (for each treatment, expressed relative to the value obtained for control [N] rats, which was set as 1.0). Right: Representative 2D-E subsections obtained from livers of N, HFD, and HFD-T2 (4 weeks) rats. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups; ***P < 0.05 vs. HFD-fed group. □, N; ■, HFD; ▨, HFD-T2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198093&req=5

Figure 4: T2 shifts hepatic gene and protein expression profiles toward increased lipid handling and decreased lipogenesis and gluconeogenesis. A–H: Quantitative RT-PCR analysis showing that T2 modulates gene expression in favor of lipid reduction (A), increases expression of genes involved in mitochondrial biogenesis (B), increases PPARα/δ expression (C and F), and genes involved in hepatic fatty acid oxidation (D and G), and normalizes the expression of genes involved in glucose homeostasis (E and H), after 2 weeks (A–E) or 4 weeks (F–H) of HFD-T2 treatment. Expression was normalized to that of Cyclophilin F. I and J: Proteomic analysis revealed protein profile changes toward adjustment of hepatic glucose metabolism (I) and lipid metabolism (J) to the HFD challenge. Left: Quantification of the data (for each treatment, expressed relative to the value obtained for control [N] rats, which was set as 1.0). Right: Representative 2D-E subsections obtained from livers of N, HFD, and HFD-T2 (4 weeks) rats. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups; ***P < 0.05 vs. HFD-fed group. □, N; ■, HFD; ▨, HFD-T2.
Mentions: The effects of T2 through SIRT1 clearly involve inhibition of lipogenesis and increased mitochondrial activity, through deacetylation of SREBP-1c and PGC-1α. Acetylated SREBP-1c induces transcription of critical lipogenic genes, and we found that after 2 weeks in the HFD-T2 animals, the SREBP-1c target genes ACC and fatty acid synthase (FAS) were downregulated (Fig. 4A). A third gene involved in lipogenesis, Spot 14 (S14), was also downregulated by T2, and a lipolytic gene, hepatic lipase (HL), involved in downregulation of triglycerides, was upregulated (Fig. 4A). Regarding mitochondrial biogenesis, T2 deacetylated PGC-1α but did not increase PGC-1α gene expression, and neither that of nuclear respiratory factors 1 and 2 (NRF1 and NRF2). However, those of mitochondrial transcription factor A and cytochrome oxidase subunit IV were increased in the HFD-T2 animals (Fig. 4B). Because fatty acid oxidation is known to be governed by PPARα and PPARδ in the liver, we measured the expression of PPARα and PPARδ, as well as the expression of a number of known PPARα/δ target genes. PPARs were targets of both AMPK and SIRT1, and gene expression was measured at both the 2-week time point (when only SIRT1 activity was increased) and the 4-week time point (when both SIRT1 and AMPK activities were increased). The PPARα/δ target genes were as follows: CPT1a and CPT2 (each involved in mitochondrial fatty acid uptake), acyl-CoA oxidase (AOX; a key enzyme in peroxisomal fatty acid oxidation), uncoupling protein 2 (UCP2; a PPAR target gene that is not translated into protein in the liver), and mitochondrial thioesterase I (MTE I; involved in mitochondrial lipid handling). In addition, because glucose tolerance was significantly ameliorated in HFD-T2 rats, we measured the expression of key genes in glucose homeostasis, which have been shown to be targets of AMPK and SIRT1. These key genes were as follows: phosphoenolpyruvate carboxykinase (PEPCK; which converts oxaloacetate to phosphoenolpyruvate and carbon dioxide), liver pyruvate kinase (LPK; a glycolytic enzyme that converts phosphoenolpyruvate to pyruvate), and glucose-6-phosphatase (G6Pase; which converts glucose-6-phosphate to glucose, which is then released from the hepatocyte). At 2 weeks, HFD-T2 rats displayed (vs. HFD rats) the following: 1) increased expression of PPARα, but not of PPARδ (Fig. 4C); 2) significant upregulation of the expression of CPT1a and CPT2 (Fig. 4D); and 3) significant reductions in the expression of LPK and G6Pase (Fig. 4E). At 4 weeks (again vs. HFD animals), HFD-T2 animals displayed significantly increased expression of both PPARα and PPARδ (Fig. 4F) and of all the above-mentioned PPARα/δ target genes (Fig. 4G). The expression of LPK and G6Pase were downregulated (as at 2 weeks), and that of PEPCK was still unaltered by T2 (Fig. 4H). Subsequently, we performed a high-resolution differential proteomic analysis on livers from N, HFD, and HFD-T2 rats. From the spots showing differential expression among the three analyzed groups, nine protein spots were identified and selected as proteins involved in glucose or lipid metabolism (Fig. 4I and J and Table 1). T2 treatment 1) prevented the induced elevations in glycolytic (LPK) and gluconeogenic (PEPCK, fructose-1,6-bisphosphatase 1 [FBPase1], and isocitrate dehydrogenase [IDH]) enzymes (Fig. 4I and Table 1), 2) had a strong positive impact on the expression of enzymes involved in fatty acid oxidation (enoyl CoA hydratase [ECH]), and 3) had normalizing effects on lipogenic proteins (carbonic anhydrase 3 [CA3] and glycerol-3-phosphate dehydrogenase [GPDH]) (Fig. 4J and Table 1).

Bottom Line: T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation.At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated.Proteomic analysis of the hepatic protein profile supported these changes.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy.

ABSTRACT

Objective: High-fat diets (HFDs) are known to induce insulin resistance. Previously, we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented gain in body weight and adipose mass. Here we investigated whether and how T2 prevented HFD-induced insulin resistance.

Research design and methods: We investigated the biochemical targets of T2 related to lipid and glucose homeostasis over time using various techniques, including genomic and proteomic profiling, immunoblotting, transient transfection, and enzyme activity analysis.

Results: Here we show that, in rats, HFD feeding induced insulin resistance (as expected), whereas T2 administration prevented its onset. T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation. At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated. Proteomic analysis of the hepatic protein profile supported these changes.

Conclusions: T2, by activating SIRT1, triggers a cascade of events resulting in improvement of the serum lipid profile, prevention of fat accumulation, and, finally, prevention of diet-induced insulin resistance.

Show MeSH
Related in: MedlinePlus